Recently new biophysical, chemical and biophysical cytochemical methods, using the fluorescent molecular probe acridine orange (AO), coupled with high resolution analytical biochemistry have been applied to the glycosaminoglycans (GAG, in general, and heparin, in particular. Preliminary results, using heparin from commercial sources as starting material and extracts from the Furth murine mastocytoma in solid and ascitic form, show that the complexes of AO with heparin: (1) can identify heparin in mixtures and in cells; (2) can differentiate various heparins from each other; (3) can determine molecular parameters, thermodynamic binding constants, and changes in conformation; and (4) can make such determinations in individual living cells. The biophysical methods employed are based primarily on fluorescence spectroscopy, (microspectrofluorophotometry) optical rotary dispersion (microspectropolarimetry), both employing the microscope. It is proposed to apply this battery of highly sensitive absolute qualitative and quantitative methodologies to continue studying heparin with a view to correlating its basic chemistry in biochemical and physical chemical terms with its anticoagulant activity. The various heparins and their interactions with ions, biopolymers, and cells will be investigated for the underlying chemistry and the results related to biological activity such as anticoagulation potency. Clues to other aspects of the biological role of heparin will be sought. One possible result of this research is the development of reproducible standard heparins with constant biological activity and biophysical/biochemical characterization sufficient for this purpose.